In recent years, a variety of peptides has attracted attentions as a drug candidate or research tool. There have been various attempts to develop a peptide library and screen peptides having affinity with a target substance.
As a method of artificially constructing a peptide library, a method using chemical synthesis, a method using a biosynthetic enzyme of a secondary metabolite, and a translation synthesis system, and the like have been employed conventionally.
It is however difficult, in the method using chemical synthesis, to increase the diversity of a library. In addition, it takes time for screening or analyzing the relationship between the structure of a compound and activity.
The method of using a biosynthetic enzyme of a secondary metabolite, on the other hand, enables rapid and easy construction or chemical conversion of a precise backbone that is difficult to be achieved by the chemical synthesis method. Since enzymes have substrate specificity, however, kinds of compounds that can be synthesized are limited. This method is therefore not suited for use in the construction of a compound library with highly diverse kinds of molecules.
When a translation system is used, a peptide library rich in diversity can be constructed in a short time by constructing an mRNA library and translating it in one pot. By using this system in combination with an mRNA display method or the like, both a peptide selected by screening and information on a nucleic acid encoding such peptide can be obtained simultaneously so that the genotype and the phenotype of the selected peptide can be related to each other easily. Despite the fact that synthesis of a peptide library using such a translation system has many advantages, it can only produce compounds consisting of peptidic backbone.
In screening using a library, identification of a compound inhibiting a target substance having protease activity is often required. The library of peptidic compounds is however cleaved by protease, and thus compounds inhibiting the activity of a target substance cannot be screened efficiently.
Each peptide of the peptide library may be modified in vitro with a post-translational modification enzyme, but an enzyme having a desired activity does not always have activity in vitro. In addition, the expressed peptide library should be purified prior to the reaction with an enzyme, and investigation of substrate specificity of the enzyme is also required. It is therefore not easy to obtain a library comprised of peptides having a desired structure.
A library in which the presence or absence, or degree of modification of each member cannot be identified is inferior in usefulness because it eventually requires correlation analysis between structure and activity, as in the chemical synthesis system.
Patellamide produced by Prochloron didemni, that is, endozoic algae of sea squirt is a low molecular cyclic peptide which is presumed to have various physiological activities and it is biosynthesized via a unique pathway with products of a pat gene cluster consisting of patA to patG. The pat gene cluster and biosynthesis pathway of patellamide are schematically shown in FIG. 15.
In this biosynthesis, PatE peptide which is a patE gene product serves as a precursor. Since the patE gene has a hypervariable region (cassette domain), the product of it constructs a natural combinatorial library.
The PatE peptide has, on both sides of the cassette domain, a recognition sequence by a post-translational modification enzyme. PatA, PatD, and PatG serve as the post-translational modification enzyme. Pat D introduces an azoline backbone into Cys, Ser, and Thr in the cassette of PatE and converts Cys to a thiazoline backbone and Ser and Thr into an oxazoline backbone.
PatE cleaves the recognition sequence at the N-terminus side of the cassette domain of the PatE.
PatG is composed of two domains. An oxidase domain on the N-terminus side converts an azoline backbone introduced by PatD into an azole backbone, that is, converts a thiazoline backbone into an azole backbone. A peptidase domain on the C-terminus side macrocyclizes PatE, while cleaving the recognition sequence on the C-terminus side of the cassette domain of PatE.
With regard to the cassette domain of the above-mentioned natural PatE, sequences shown in the following table are described in M. S. Donia et al. (Non-patent Document 1).
[Table 1] is provided as FIG. 22.
TABLE 2COMPOUNDCODING SEQUENCEpatellamide family:patellamide C (E1I, E2I)V T A C I T F Cpatellamide A (E1II)I T V C I S V Cpatellamide B (E4I, E5I)L T A C I T F Cnew compound 1 (E6I)V A A C I T F Cnew compound 2 (E7I)L T T C I T F Cnew compound 3 (E8I)L T A C V T F Cnew compound 4 (E9II)I T V C I T V Cnew compound 5 (E10I)L A A C I T F Cnew compound 6 (E11I)L T A C I T L Cnew compound 7 (E12II)I T V C I S A Cnew compound 8 (E13II)S T V C F T V Cnew compound 9 (E15I)V T A C I A F Cnew compound 10 (E16I)V T A C I T S Cnew compound 11 (E17I)V T A C I T L Cnew compound 12 (E18I)V T T C I T F Cnew compound 13 (E20I)V T A C T T F Culithiacyclamide family:ulithiacyclamide (E2II)C T L C C T L Cnew compound 14 (E14II)C T L C C T L Rnew compound 15 (E19II)C I L C C T L Cnew compound 16 (E21II)C T L C C A L Cnew compound 17 (E22II)C T L C C T V Cnew compound 18 (E23II)C T L C C T F Cnew compound 19 (E24II)C T V C C A V Cnew compound 20 (E25II)C T L C Y T L Clissoclinamide family:lissoclinamide 2/3 (E3I)  A C F P T I Clissoclinamide 4/5 (E3II)  F C F P T V Cnew compound 21 (E26II)  L C F P T V Cnew compound 22 (E27II)  F C V P T V Cnew compound 23 (E28II)  F C F P A V Cnew compound 24 (E29II)  F C L P T V Cpatellamide Aulithiacyclamidelissoclinamide 2/3
The corresponding sequence numbers of Table 2 from top to bottom are: SEQ ID NOS: 60, 348-376, respectively.
These tables show that sequences of natural cassette domains have following similarities: (i) they have 7 or 8 residues, (ii) they tend to have Ser/Thr/Cys to be modified at 2, 4, 6, or 8 positions from the N-terminus of the cassette domain, (iii) the residues (Ser, Thr, and Cys) to be modified are not adjacent to each other in most cases, and (iv) many of the residues other than Ser, Thr, and Cys are hydrophobic residues such as Val, Ala, Ile, Phe, and Leu.
These similarities were presumed to be necessary for becoming a substrate of PatD or PatG, a post-translational modification enzyme. It is however not known which residue of Ser, Thr, and Cys has been modified or not modified and substrate specificity of PatD and PatG has not been elucidated yet.